DESCRIPTION (Adapted from abstract): Rhodopsin is the prototypical G protein-coupled receptor. The G-protein receptors are thought to have a common architecture built around seven transmembrane helices. Ligand binding in most GPCRs or retinal isomerization in the case of rhodopsin triggers a protein conformational change that allows binding of G proteins to the cytoplasmic surface of the receptor. The high resolution structure is not known for any member of this large receptor family. Moreover, there is no consistent mechanism for how ligand binding activates a GPCR. The pharmaceutical importance of this class of receptor is revealed in the estimate that 60% of the targets for all drugs sold today are GPCRs. In the last few years the investigators have made progress in establishing the structure of the retinal binding site in rhodopsin and how retinal isomerization triggers rhodopsin activation. Recent progress in the expression and purification of rhodopsin containing 13C and 15N labels has opened a new route for structural studies. Three specific aims form the core of this proposal. Structural studies are proposed for determining the retinal conformation and how it is packed in the protein interior. These studies involve establishing the orientation of the retinal in the protein binding pocket and the contact points between specific retinal carbons and residues in the protein binding site. NMR measurements are proposed for establishing the structure changes involved in rhodopsin activation. Glutamic acid, histidine and the retinal PSB linkage are thought to change protonation state upon formation of metarhodopsin 11. The investigators plan to measure changes in the protonation state of these residues during the rhodopsin photoreaction. Changes in retinal and protein structure will also be determined by measuring distances between specific retinal and protein isotopic labels. NMR studies are proposed for determining the structure and interactions of the C-terminal peptide of the a-subunit of transducin bound to metarhodopsin 11. Intrapeptide distances will be measured to determine the peptide conformation. Specific peptide-receptor distances will be measured by engineering unique sites into the loops and C-terminus of rhodopsin. Together these studies lay the foundation for establishing ligand conformation and binding contacts in the family of GPCRs, the long term objective of the proposed research.